Gas Chromatograph-Combustion-Continuous Counting System for Analysis of Microgram Amounts of Radioactive Metabo1ites R. 0. Martin Department of Biochemistry, Unicersity of Saskatchewan, Saskatoon, Sask., Canada
The total effluent of a gas chromatograph is passed over copper oxide at 800 O C followed by measurement of the carbon dioxide formed with a microthermistor detector. The radioactivity of the carbon dioxide after mixing with propane is measured by a simple proportional counter. There are no split flow corrections, and errors in specific activities caused by combustion variability are minimized. This approach is useful over a large range of organic compounds up to molecular weights near 400. The mass detector is sensitive to less than 0.lFg and shows a linear response over a 2000-fold concentration range. 200 to 100,000 dpm of 14C can be detected in a given peak. Both mass detector and proportional counter operate without contamination at ambient temperature.
CONTINUOUS MEASUREMENT of the radioactivity in compounds eluted from a gas chromatograph has been the subject of several reviews (1-3). Our interest in the problem began with the need for a sensitive method for determining the specific activities of microgram amounts of radioactive alkaloids obtained from a few grams of plant material after short periods of photosynthesis in an atmosphere of 14C02 (4). This earlier work (4) utilized a triode argon mass detector operated at 300 "C,followed by combustion and proportional counting of the total effluent. Fouling of the high temperature detector was frequent in that system. Although Martin and Smart ( 5 ) utilized combustion of organic compounds to COz and HzO followed by thermal conductivity detection of the COz, previously published approaches to the counting problem utilized combustion of the GLC effluent following the mass detector [usually a low sensitivity thermal conductivity detector was used (1-4)], The present system uses combustion of the total effluent of the GCfollowed b y both mass and acticity measurement at ambient temperature. The large, fixed thermal conductivity difference between helium and C02 coupled with a rugged, small volume microthermistor detector results in stable, reproducible high sensitivity mass detection. Details are given for a n inexpensive, easily assembled proportional counter having excellent characteristics. Neither detector has required cleaning after nearly a year of regular use. EXPERIMENTAL
Reagents. Octadecane-lJ4C obtained from New England Nuclear Corporation was purified by GLC. The phenylalanine-2- and-U- I4C were kindly supplied by Dr. Underhill of the National Research Council. Trimethylsilyl (TMS)
(1) F. Cacace, Nucleonics, 19,45 (1961). (2) H. J. Dutton, J . Amer. Oil Chem. SOC.,38, 631 (1961). (3) F. Drawert and 0. Bachmann, Angew. Chem., 75, 717 (1963). (4) R. 0. Martin, M. E. Warren, and H. Rapoport, Biochemistry 6 , 2355 (1967). (5) A. E. Martin and J. Smart, Nature, 175, 422 (1955).
Figure 1. GLC-combustion-flow counting system derivatives of amino acids were prepared just prior to use by the method of Klebe (6). Apparatus. A block diagram of the complete system is shown in Figure 1. All glass t o metal seals are made with silicone rubber O-rings in place of the Swagelok front ferrule, A Micro-Tek M T 220 DP unit supplied the chassis, oven, temperature, and gas flow controls. A solvent venting valve, 4 , is provided at the sample column, 2 , exit port by silver soldering a 3.5-inch length of heavy-wall, l/g-inch stainless steel tube into a hole drilled through the left side of the extreme left exit fitting mounting flange. A toggle valve permits its rapid, complete opening and closing. A Sargent Model S-36400 combustion furnace, 5 , rests on its side o n a platform on top of the oven frame. The center of the furnace proper is located directly over the sample exit port. Both hinge flanges of the furnace cover are cut back flush, and the cover is rehinged to prevent obstruction of the injection port, I . Combustion tubes of an inverted "L" form, 23 cm tall and 10 cm deep, are of 6-mm Vycor tubing. A cone of stainless steel gauze supports the 20-30 mesh copper shot, 6. Loose plugs of glass wool retain the 10-20 mesh anhydrous MgC104, 8. The lower end of the combustion tube is secured directly to the column exit port. The upper end joins a gas-tight 3-way valve, 9, (Hamilton CO.). Both the microthermistor detector, IO, (Carle Instruments Co., Model 100 Standard micro detector system) and the counter tube, 13, are mounted with bunsen clamps to a verticle rod screwed to the furnace chassis. The reference inlet of the Carle detector joins the reference column, 3, filled with uncoated support uia a length of '/la-inch stainless tubing, 7; I 1 is the Carle detector control unit. A short ( 6 ) J. F. Klebe, H. Finkbeiner, and D. M. White, J . Amer. Chem. SOC.,88, 3390 (1966). VOL. 40, NO. 8, JULY 1968
1197
Table I. Mass Detector Sensitivity, Linearity and Precision for Octadecane" ,dinjected Pg Area Deviation 2 0.2 1.5 &5 8 0.8 5.6 *3 20 2.0 12.0 1 5 2 2.0 9.0 It5 40 4.0 22.0 1 2 8 8.0 64.0 *l 20 20 160 f 2 2 20 145 f 5 40 40 320 *2 8 80 500 1 2 20 200 1280 1 2 40 400 2700 f l a Column: borosilicate glass l/4-inch X 4 ft, 5 % QF-1 on 60-70 mesh HMDS treated Chromasorb, 150 "C; He, 50 ml/min. Three concentrations of 0.1, 1.0., and 10 mg/ml in heptane were used.
Table 11. Analysis of Pheny1alanine-2-l4C (TMS)" dpm pl injected I.rg injected measured 1 0.1 140 170 3 0.3 420 381 1 0.4 558 520 6 0.6 840 960 3 1.2 1670 1280 6 2.4 3340 2950 1 2.5 3500 3000 3 7.5 10400 11040 6 15.0 21000 24300 a Column conditions same as for Table I; Propane = 12 ml/min; He = 40 ml/min; high voltage, 2.3 kV; background: 60 cpm; furnace 800 "C.
length of 1/2-inchglass tube filled with 10-20 mesh indicator soda lime, 14, traps any 14C02. A simplified and improved counter similar t o previously described ones (3, 7, 8) is shown in Figure 2. Dimensions are for a 16.5-ml counter. The tube after assembly is always kept in a vertical position. Propane flow control requires a fine metering valve. Directly attached to the U G 560/U Connector, B, Figure 2, is a Nuclear Chicago Model 8765 preamplifier, 12. A Model 8735 digital integrator and a 8733 rate meter power supply, 15, are mounted at the bottom of the GC chassis. The digital printer is 16. A simultaneous monitoring of the mass detector and ratemeter outputs are on a Hewlett-Packard Moseley 7128A dual pen recorder, 17. Operation and Calibration. GENERATION OF COPPER OXIDE. With the column oven heat and carrier gas on, and the furnace a t 800 " C the solvent vent is opened and oxygen (ca. 30 ml/min) is passed via the 3-way valve through the combustion tube only, for 1-2 minutes. Both valves are ' then closed. Counter Plateau and Efficiency Determination. With the helium flow set at about 40 ml/min and the propane at 15 rnl/min, the high voltage is turned on and the plateau determined with a n external I3'Cs source. As a standard, 4-8 pl aliquots of a solution of ~ c t a d e c a n e - l - ~in~ cheptane (ca. 350 dpm/kl) are injected; the solvent vent is opened immediately, then closed after one minute. From the recorded counts under the octadecane peak, the dynamic efficiency of the counter is determined by the relation (7) R. Wolfang and F. S . Rowland, ANAL.CHEM.,30, 903 (1958). (8) A. T. James and E. A. Piper, J. Chromatogr., 5,265 (1961). 1198 *
ANALYTICAL CHEMISTRY
Astd
where Natdequals the net recorded counts for the octadecane standard (650-1300 counts actually taken) and Astd equals the absolute activity (dpm) of the octadecane standard (1400-2800 dpm injected). Mass Detector Calibration. Known amounts of desired mass standards are injected, and the areas of the resulting mass peaks are determined by triangulation. Warm-up time, including counter calibration from a cold start is less than an hour. Analysis of Sample. The absolute activity in a given peak is determined by
where N and A are defined the same as for the standard. Sample volumes from 1-50 pl have been used. In all cases, the solvent vent should be opened just prior to injection and closed about one minute after injection of sample; the length of the venting period is determined by the volume of sample injected and the volatility of the solvent. RESULTS AND DISCUSSION
Mass Detector Performance. Noise level is less than 0.05 mV or 1 % of full scale o n the most sensitive range used. Isothermal base line drift is about 0.3 mV/hr or 8% of full scale on the most sensitive range used. The Carle microthermistor detector is operated at only liz0its maximum sensitivity. The small diameter reference flow tube through the furnace is essential for stable operation. The reference GLC column is packed with uncoated support material t o avoid any liquid phase condensation inside the reference tube. Programmed temperature operation does not result in objectionable base line shift. Sensitivity limits are in the order of 0.05 pg for C,N,O containing compounds such as those tested here. Dependence o n the elemental composition of different compounds has not been examined in any systematic way. Because known compounds are usually injected t o determine retention times, they are utilized as mass standards. The possibility of increasing the sensitivity, by converting the water formed on combustion into acetylene, was tested by substituting calcium carbide for the magnesium perchlorate. The dynamic range of the detector was determined by injecting increasing amounts of octadecane from 0.2 to 400 pg. The results of Table I show a linear response to within = t 5 % over this 2000-fold concentration range. Larger masses were not tested because of serious column overloading. Reproducibility varies from ~ t 5 %up to 4 pg and =k2% for greater amounts. Simplicity of the mass detector system is obvious. I t is compact, operates at ambient temperature; plumbing lengths and insulation after combustion area are unimportant, and cleaning is rarely required. I n order to obtain reproducible results, two things are necessary: first, venting of the solvent after injection t o prevent rapid depletion of the copper oxide, and second, regeneration of the copper oxide about every 8 to 12 samples (as noticed by a decrease in detector response). An increasingly erratic base line indicates an obstructed copper oxide column. When this happens, the column is discarded because fusion of the copper beads makes cleaning difficult. Counter Performance. The plateau begins a t about 2100 V, is 500-700 V wide with a slope of 25 counts/100 V when using a n external 1 3 7 0 source and a helium propane ratio of be-
c
a
e< +C
-A
D-
-F
EH-
Figure 2. Proportional gas flow counter A . Stainless-steeltube, 0.75 x 0.40 X 8 inches honed inside to mirror finish B. Amphenol UG 560/U receptacle with base locking ring removed C. Rubber O-ring D. 0.002-inch stainlesssteel wire E, Cylindrical weight made of Teflon (Dupont) and grooved along outside F. 0.80 Brass nut G . l/8-inch heavy wall stainless-steeltube H . Tapped for 3/8-inch pipe thread
TEMP.(C*)
MIMJTES
Figure 3. Extracts of biological material after feeding "COZ A . O~tadecane-l-~~C as received from supplier. 1. Vent opened; 2. Vent closed; 3. Octadecane; 4. Unknown B. Lycopodium Alkaloids 1. Lycopodine; 2. Alkaloid L-20 (?); 3. Lycoclavine (?) C. Yeast Amino Acids 1. Aspartic Acid; 2. Glutamic Acid; 3. Saccaropine (?) D. Conium Alkaloids 1. N-Methyl coniine; 2. Coniine; 3. yconiceine
tween 3 and 4 : l . Background for the unshielded detector is about 60 cpm at ambient temperature. Background for a newly assembled counter or one which has been standing without carrier gas flow, may be 200-300 cpm for an hour or so. Dynamic efficiencies with total flow rates of 52 ml/min and a counter volume of 27 ml are ca. 40% which calculates to a static efficiency of 80% from the relation:
V eatatic
= eavnamtc X -
f
(3)
where V is the counter volume in ml and f is the total flow through the counter in ml/min. Octadecane-l-14C was chosen in place of the more volatile toluene- or hexane-I4C for standardizing the counter, because the main solvent (hexane) peak could be vented to avoid passing through the combustion system large mass samples which rapidly deplete the copper oxide. Table I1 shows the results of a series of duplicate injections of varying amounts of phenylalanine-2-14C (as TMS derivative) of known specific activity over a 150-fold activity range. The absolute activities of the individual samples injected were known to only *5-10%. This coupled with counting statistics results in the large deviations. The mass area values are not shown as certain problems of mass detector stability had not been solved at the time.
Table I11 shows the results of a series of quadruplicate samples of phenylalanine-U-14C (as TMS derivative). The low value here could be caused by incomplete combustion of the aromatic ring, although an even greater discrepancy would have been expected if this were the case. Other possibilities are incomplete conversion to the TMS derivative, or the presence of a nonvolatilized radio impurity in the sample. No other activity peaks were detected. The last column shows increase in sensitivity of mass measurement when calcium carbide is substituted for magnesium perchlorate as drying agent. General Utility of the System. Representative recorder traces are shown in Figure 3 from the analysis of a number of radioactive biological extracts. Column conditions for all four samples are the same as for Table 11, except that for C and D,the column packing was 1 % carbowax, 5% KOH on Chromosorb W. In all cases the lower, erratic line is the radioactivity trace. The octadecane sample ( 3 A ) provides a valuable lesson--i.e., never to take the radiochemical purity of a sample for granted. The identity of the second radioactivity peak is not known, but accounted for nearly 4 0 z of the total sample activity. Figure 3B shows the radioactivity distribution in a 1/50aliquot in the alkaloids from 20 grams of fresh Lycopodium plants after a 2-hour exposure to 1 m C of W02. Figure 3C represents 1/20 of the total amino acids (as T M S derivative) from 1 gram of yeast after incubation with 10 VOL 40, NO. 8, JULY 1968
1199
Table 111. Analysis of Phe~~ylalanine-U-~~C (TMS)"
injected Pg dpm expecteda 3 7.5 4900 6 15.0 9800 = Conditions were the same as for Table 11. Determined by scintillation count. p1
MCof N a H L 4 C 0 8for 40 minutes. Figure 30 represents 1/100 of the total alkaloid extract of 3 grams of fresh Conium plants after 2 hours in a n atmosphere containing 2 m C of I4CO2. The improved resolution and accuracy of the digital data system employed over the conventional ratemeter display has been discussed by several authors (9). A plot of the (9) B. Kawain and F. V. Huston, Nucleonics, 22, 86 (1964).
dpm by GLC 4,600 9,ooO
Mass area 5 . 0 i 0.3 9.0 f 0.2
Mass area with CaC 6.25 f 0.3 12.5 rt 0.5
digital data for the first triplet of radioactivity peaks in Figure 3 D shows these peaks to be almost completely resolved. ACKNOWLEDGMENT
The author thanks Mrs. E. Welgan for her technical assitance.
RECEIVED for review December 18, 1967. Accepted April 22, 1968. This work was supported financially by the National Research Council.
Liquid-Liquid Extraction of Nickel with Longchain Amines from Aqueous and Nonaqueous Halide Media T. M. Florence and Yvonne J. Farrar Analytical Chemistry Section, Australian Atomic Energy Commission, Lucas Heights, N . S. W., Australia
A detailed study has been made of the extraction of nickel from concentrated halide salt solutions by longchain amines. Solvent extraction data and spectral evidence show that nickel is present in the organic amine phase as the highly colored, tetrahedral complex NiX42-. With correct conditions high distribution ratios for nickel can be achieved, extraction increasing in the order CI- < Br- < I - a t constant halideconcentration. Amine diluent, halide cation, and free acidity have a strong effect on the nickel distribution ratio, the highest ratios being obtained when the amine is dissolved in cyclohexane, lithium halide salts are used, and the free acidity i s low. The concentration of halide necessary for extraction of nickel can be greatly reduced if the lithium salt is dissolved in a nonaqueous solvent and the extraction carried out with two immiscible organic layers. Examples of nonaqueous liquidliquid extractions of this type are given in which nickel is extracted from solutions of lithium halides in anhydrous methanol.
traction of nickel from 13.OM lithium chloride with tri-noctylamine in toluene, and showed that nickel was present in the organic phase as the tetrahedral NiC142- species. The poor extraction of nickel by long-chain amines is apparently due to the reluctance of Ni(I1) to form anionic complexes in aqueous halide solutions. A recent investigation of the polarography of nickel in concentrated halide media (6) showed that the predominant nickel species in saturated aqueous lithium chloride is Ni(H20)rC1+, but that the tetrachloronickelate(I1) ion, NiC142-, is produced in methanolic solutions of lithium chloride. Both high chloride activity and low free water activity were necessary for the formation of the nickel chloride complexes. Previous work has shown that NiCld2- can exist in molten salts (7-9), molten salt hydrates (IO), and various organic solvents (11-16). Very recently, Scarrow and Griffiths (17) detected NiC142- in hot, concen-
SEVERALSURVEYS (1-3) on the extraction of metals by longchain amines have shown that nickel is extracted t o a negligible extent from hydrochloric acid solutions. Seeley and Crouse ( 4 ) made a systematic study of the extraction of 63 metal ions from both hydrochloric acid and lithium chloride media, using primary, secondary, tertiary, and quaternary amines. Little or no extraction of nickel was observed. Lindenbaum and Boyd (3, however, reported significant ex-
(6) T. M. Florence, Australian J. Cliem., 19, 1343 (1966). (7) D. M. Gruen and R. L. McBeth, J . Phys. Clzem., 63, 393 (1959). (8) G. P. Smith, C. H. Liu, and T. R. Griffiths, J. Am. Chem. SOC., 86, 4796 (1964). (9) C. A. Angell and D. M. Gruen, J. Pliys. Chem., 70, 1601 (1966). (10) C. A. Angell and D. M. Gruen, J. Am. Chem. Soc., 88, 5192 (1966). (11) W. D. Beaver, L. E. Trevorrow, W. E. Estill, P. C. Yates, and T. E. Moore, J. Am. Clzem. Soc., 75,4556 (1953). (12) L. I. Katzin, Nature, 182,1013 (1958). (13) N. S. Gill and R. S. Nyholm, J. Chem. SOC.,1959, 3997. (14) D. M. L. Goodgame, M. Goodgame, and F. A. Cotton, J . Am. Chem. SOC.,83, 4161 (1961). (15) D. W. Meek, D. K. Straub, and R. S. Drago, ihid., 82, 6013 (1960). (16) D. F. C. Morris and D. N. Slater, J . Inorg. Nucl. Chem., 27, 250 (1965). (17) R. K. Scarrow and T. R. Griffiths, Chem. Communs., 1967, 425.
(1) C. F. Coleman, Nucl. Sci. Eng., 17, 274 (1963). (2) C . F. Coleman, C. A. Blake, and K. B. Brown, Talanfa, 9, 297 (1962). (3) T. Ishimori, Japan Ar. Energy Res. Inst., Rept. JAERI-1047 (1963). (4) F. G. Seeley and D. J. Crouse, J . Chem. Eng. Data, 11, 424 (1966). (5) S. Lindenbaum and G. E. Boyd, J. Phys. Clzem., 67, 1238 (1963). 1200
0
ANALYTICAL CHEMISTRY
